Novel Retinitis Pigmentosa Treatment

ABSTRACT

An isolated or purified antisense oligomer for modifying pre-mRNA splicing in the CNOT3 gene transcript or part thereof.

TECHNICAL FIELD

The present invention relates to the use of antisense oligomers totreat, prevent or ameliorate the effects of retinitis pigmentosa.

BACKGROUND ART

Retinitis pigmentosa (RP) is degenerative eye disease that causes severevision impairment due to the progressive degeneration of the rodphotoreceptor cells in the retina; most cases of RP are inherited. Thisform of retinal dystrophy manifests initial symptoms independent of age;thus, RP diagnosis occurs anywhere from early infancy to late adulthood.RP is a rare disease that affects approximately one out of 4000individuals (more than 1.5 million people worldwide). Of the familial RPcases, 30%-40% show autosomal-dominant genetic inheritance (Hartong etal. 2006). There is currently no treatment for RP.

RP is caused by mutations in more than 50 genes. Heterozygous mutationsin the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP).In some cases, such mutations display incomplete penetrance, whereincertain carriers develop retinal degeneration while others have nosymptoms at all. Asymptomatic carriers are protected from the disease bya higher than average expression of the PRPF31 allele that is notmutated.

Expression of the PRPF31 gene is controlled by the Ccr4-Not deadenylasecomplex. Ccr4-Not is a nine-subunit protein complex that is a masterregulator of translation and mRNA stability in eukaryotic cells. Thecore CCR4-Not complex consists of Ccr4p, Caf1p, five Not proteins(CNot1-CNot5), Caf40p, and Caf130p in yeast.

At the present time, AAV mediated gene replacement and CRISPR/Cas9 geneediting are being explored as therapies for retinal disease. While thecoding sequence of PRPF31 is within the capacity of AAV vectors, theconsequences of unregulated or over-expression of PRPF31 are unknown. Inaddition, it is not known if ocular viral mediated gene therapies couldbe re-administered, since seroconversion as a consequence of intraocularviral vector injection has been reported. Furthermore, CRISPR/Cas9 genecorrection will require a different product for each family's PRPF31mutation. In addition, both the gene replacement and gene editingapproaches require subretinal injection of viral vectors to achieveadequate transfection.

There is a need to provide new treatments or preventative measures forretinitis pigmentosa; or at least the provision of methods to complimentthe previously known treatments.

The present invention seeks to provide an improved or alternative methodfor treating, preventing or ameliorating the effects of retinitispigmentosa.

The previous discussion of the background art is intended to facilitatean understanding of the present invention only. The discussion is not anacknowledgement or admission that any of the material referred to is orwas part of the common general knowledge as at the priority date of theapplication.

SUMMARY OF INVENTION

Broadly, according to one aspect of the invention, there is provided anisolated or purified antisense oligomer for modifying pre-mRNA splicingin the CNOT3 gene transcript or part thereof. Preferably, there isprovided an isolated or purified antisense oligomer for inducingnon-productive splicing in the CNOT3 gene transcript or part thereof.

For example, in one aspect of the invention, there is provided anantisense oligomer of 10 to 50 nucleotides comprising a targetingsequence complementary to a region near or within an intron of the CNOT3gene transcript or part thereof. In another aspect of the invention,there is provided an antisense oligomer of 10 to 50 nucleotidescomprising a targeting sequence complementary to a region near or withinan exon of the CNOT3 gene transcript or part thereof.

Preferably, the antisense oligomer is a phosphorodiamidate morpholinooligomer. Preferably the antisense oligomer has a modified backbone.

Preferably, the antisense oligomer is selected from the group comprisingthe sequences set forth in Table 1.

Preferably, the antisense oligomer is selected from the list comprising:SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18,27, 30, 34, 35, 64 and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30,34 and 64.

The antisense oligomer preferably operates to induce skipping of one ormore of the exons of the CNOT3 gene transcript or part thereof. Forexample, the antisense oligomer may induce skipping of exons 3, 8, 9, 12and/or 17.

The antisense oligomer of the invention may be selected to be anantisense oligomer capable of binding to a selected CNOT3 target site,wherein the target site is an mRNA splicing site selected from a splicedonor site, splice acceptor sites, or exonic splicing elements. Thetarget site may also include some flanking intronic sequences when thedonor or acceptor splice sites are targeted.

More specifically, the antisense oligomer may be selected from the groupcomprising of any one or more of SEQ ID NOs: 1-74, more preferably SEQID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67 even morepreferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64 and/or the sequences setforth in Table 1, and combinations or cocktails thereof. This includessequences which can hybridise to such sequences under stringenthybridisation conditions, sequences complementary thereto, sequencescontaining modified bases, modified backbones, and functionaltruncations or extensions thereof which possess or modulate pre-mRNAprocessing activity in a CNOT3 gene transcript In certain embodiments,antisense oligomers may be 100% complementary to the target sequence, ormay include mismatches, e.g., to accommodate variants, as long as aheteroduplex formed between the oligonucleotide and target sequence issufficiently stable to withstand the action of cellular nucleases andother modes of degradation which may occur in vivo. Hence, certainoligonucleotides may have about or at least about 70% sequencecomplementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, betweenthe oligonucleotide and the target sequence.

The invention extends also to a combination of two or more antisenseoligomers capable of binding to a selected target to induce exonexclusion in a CNOT3 gene transcript, including a construct comprisingtwo or more such antisense oligomers. The construct may be used for anantisense oligomer-based therapy.

The invention extends, according to a still further aspect thereof, tocDNA or cloned copies of the antisense oligomer sequences of theinvention, as well as to vectors containing the antisense oligomersequences of the invention. The invention extends further also to cellscontaining such sequences and/or vectors.

There is also provided a method for manipulating splicing in a CNOT3gene transcript, the method including the step of:

-   -   a) providing one or more of the antisense oligomers as described        herein and allowing the oligomer(s) to bind to a target nucleic        acid site.

There is also provided a pharmaceutical, prophylactic, or therapeuticcomposition to treat, prevent or ameliorate the effects of a diseaserelated to CNOT3 expression in a patient, the composition comprising:

-   -   a) one or more antisense oligomers as described herein and    -   b) one or more pharmaceutically acceptable carriers and/or        diluents.

The composition may comprise about 1 nM to 1000 nM of each of thedesired antisense oligomer(s) of the invention. Preferably, thecomposition may comprise about 10 nM to 500 nM, most preferably between1 nM and 10 nM of each of the antisense oligomer(s) of the invention.

There is also provided a method to treat, prevent or ameliorate theeffects of a disease associated with CNOT3 expression, comprising thestep of:

-   -   a) administering to the patient an effective amount of one or        more antisense oligomers or pharmaceutical composition        comprising one or more antisense oligomers as described herein.

There is also provided the use of purified and isolated antisenseoligomers as described herein, for the manufacture of a medicament totreat, prevent or ameliorate the effects of a disease associated withCNOT3 expression.

There is also provided a kit to treat, prevent or ameliorate the effectsof a disease associated with CNOT3 expression in a patient, which kitcomprises at least an antisense oligomer as described herein andcombinations or cocktails thereof, packaged in a suitable container,together with instructions for its use.

Preferably the disease associated with CNOT3 expression in a patient isretinitis pigmentosa. The subject with the disease associated with CNOT3expression may be a mammal, including a human.

Further aspects of the invention will now be described with reference tothe accompanying non-limiting examples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention are more fully described inthe following description of several non-limiting embodiments thereof.This description is included solely for the purposes of exemplifying thepresent invention. It should not be understood as a restriction on thebroad summary, disclosure or description of the invention as set outabove. The description will be made with reference to the accompanyingdrawings in which:

FIG. 1 is a schematic of the CNOT3 reading frame. CNOT3 consists of 18exons and encodes a protein with 753 amino acids. FIG. 1(a) in-frameexons are depicted by rectangles, whereas exons with junctions thatinterrupt codons are shown with chevron sides (blue, red). FIG. 1(b) theCNOT3 protein is represented showing functional domains and amino acidpositions corresponding to the exons above. NAR: NOT anchored region.CS: connecting sequence. NOT box: Negative on TATA box. Exclusion ofexons 2, 3, 8, 9, 11, 12, 13, 14, 15 and 16 will all disrupt the openreading frame.

FIG. 2 is a gel image showing CNOT3 transcript products from RP11patient fibroblasts 48 hr after 2′O-Methyl phosphorothioate AOtransfection (50, 25 and 12.5 nM). Control AOs that target no knownsequence (−ve control AO) were included for comparison.

FIG. 3 is a gel image showing CNOT3 transcript products from fibroblasts48 hr after transfection with 2′O-Methyl phosphorothioate AOs, designedto induce terminal intron retention, at concentrations of 50 and 25 nM.

FIGS. 4A and 4B: FIG. 4A is Pedigree 0255: 11 members with PRPF31mutation (c.267delA). FIG. 4B is Pedigree 0080: 13 affected members(c.1205 G>A). Arrow=dermal fibroblasts donated, black=patients currentlyin the CRE natural history study.

FIGS. 5A-H show skin fibroblasts from a patient with CLN3 mutationreprogrammed to pluripotency. Patient-iPSC (CLN3−/−) displayed typicaliPSC morphology and expressed pluripotency markers, including OCT4,NANOG, SOX2 and SSEA4 (A). Comparison pluripotent gene expression in 6iPSC lines by quantitative RT-PCR demonstrated similar expressionpatterns for all lines (B). Patient iPSC demonstrated trilineagedifferentiation potential. Patient iPSCs (CLN3-iPS-EB), gene correctedcontrol iPSCs (CLN3HDR-iPS-EB) and control human iPSC (WT-iPS-EB,ThermoFisher) were differentiated as embryoid bodies for 2 weeks thenscreened for expression of markers of ectoderm (PAX6, OTX1), mesoderm(SOX17, GATA4, FOX2A) and endoderm (BRACHYURY, FDGFR) lineages, as wellas pluripotency genes (OCT4, NANOG, SOX2) by quantitative RT-PCR (C).D-H: Retinal differentiation of iPSC. Retinal organoids displayed anoptic cup-like morphology, surrounded by transparent, laminated retinaltissues (D) that contained an organized apical layer of recoveringexpressing photoreceptors (E) as well as basally positioned Smi32expressing retinal ganglion cells (F). Electron microscopy ofphotoreceptor outer segments in day 170 retinal organoids (G)demonstrated similar morphology to outer segments in the developing(E120) human retina (H).

FIG. 6 shows screening of AO-induced CNOT3 exon skipping using2′-O-Methyl chemistry on PS backbone. AOs are designed to target spliceenhancer motifs of CNOT3 exons to mediate the exclusion of targetexon(s) during pre-mRNA splicing in order to mediate knockdown of CNOT3or disrupt protein function. Dermal fibroblasts were transfected for 48hr with CNOT3 AOs (2′OMe-PS chemistry) targeting exons (as indicated).RT-PCR products were separated on 2% agarose gels.

FIG. 7 shows screening of AO-induced CNOT3 exon skipping using2′-O-Methyl chemistry on PS backbone. AOs are designed to target spliceenhancer motifs of CNOT3 exons to mediate the exclusion of targetexon(s) during pre-mRNA splicing in order to mediate knockdown of CNOT3or disrupt protein function. Dermal fibroblasts were transfected for 48hr with CNOT3 AOs (2′OMe-PS chemistry) targeting exons (as indicated).RT-PCR products were separated on 2% agarose gels.

FIG. 8 shows the negative correlation between CNOT3 and PRPF31 mRNAexpression. FIG. 8(a) qRT-PCR analysis of PRPF31 and CNOT3 mRNAexpression normalised to TATA-binding protein (TBP) expression iniPSCs-derived retinal pigment epithelium from RP11, asymptomatic andhealthy (WT) individuals. RP11 and the asymptomatic subject are from thesame pedigree, both heterozygous for the PRPF31 c.1205C>A (Ser402*)mutation. The healthy subject is from an unrelated family. Expression ofCNOT3 and PRPF31 mRNA in the healthy control was set to 1. (n≥2). FIG.8(b) Dermal fibroblasts from an RP11 patient were transfected for 48 hrwith CNOT3 AOs (2′OMe-PS) targeting exon 4, 6, 7 or 10. PRPF31transcript level was analysed using qRT-PCR and normalised against TBPexpression. PRPF31 expression in cells treated with control AO at 25 nMwas set to 1 (n=1). FIG. 8(c) Dermal fibroblasts from an RP11 patientwere transfected for 48 hr with CNOT3 AOs (2′OMe-PS chemistry) targetingexon 3, 8, 9, 16 or 17. The PRPF31 transcript level was analysed usingqRT-PCR and normalised against TBP expression and is shown relative toPRPF31 expression in cells treated with a sham control AO at 25 nM(control PRPF31 expression set to 1 (n=1).

FIG. 9 shows the effect of ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107),targeting CNOT3 exon 17) synthesized as phosphorodiamidate morpholinooligomer (PMO) and transfected into RP11 iPSC-derived RPE. (A) CNOT3exon 17 skipping in cells treated with PMO alone or cell penetratingpeptide-tagged PMO (PPMO) at a concentration of 5 μM. (B) PRPF31upregulation as a consequence of CNOT3 knockdown, determined by qRT-PCRand normalised to TATA-binding protein (TBP) expression. PRPF31expression in untreated control was set to 1.

FIG. 10 shows (A) immunostaining of cilia (red) and basal body (green)in wildtype and RP11 RPE with or without antisense oligomer treatmentwith ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17).(B) The percentage of RPE cells expressing cilia, counted from >1,000cells. (C) Cilia length measurement using NIS-Elements Imaging software.Bar chart represents mean±SEM from ˜300 ciliated cells. Scale bar=10 μm.Student's t test. ***p<0.001.

DESCRIPTION OF INVENTION Detailed Description of the Invention AntisenseOligonucleotides

There is an increased vulnerability of photoreceptors to a generallyreduced splicing activity. Whilst mutations in splicing factors maycause splicing deficiency in a variety of tissues, photoreceptor cellswould be greatly affected due to their high demand for mRNA production(such as mRNA encoding rhodopsin and other phototransduction proteins).This model presumes that mRNA amounts produced by the retina greatlyexceed mRNA levels produced by other tissues.

PRPF31 (also known as hPRP31) encodes an essential pre-mRNA splicingfactor required for the assembly and recycling of the U4/U6 RNA complex.Several studies have demonstrated reduced amounts of total PRPF31 mRNAin patients with RP11, as well as delayed rate of spliceosome assemblyand pre-mRNA processing. This indicates that the disease occurs via ahaplo insufficiency mechanism. In comparison to other tissues, theretina expresses seven times more major spliceosomal small nuclear RNAs(snRNAs).

CCR4-Not transcription complex subunit 3 (CNOT3, one of the five Notproteins of the Ccr4-Not deadenylase complex) has been identified as themain modifier gene determining penetrance of PRPF31 mutations, via amechanism of transcriptional repression; modulating PRPF31 transcriptionby directly binding to its promoter. The expression level of thewild-type PRPF31 allele determines whether carriers of the mutatedPRPF31 allele are symptomatic. In asymptomatic carriers, CNOT3 isexpressed at low levels, allowing higher amounts of wild-type PRPF31transcripts to be produced and preventing manifestation of retinaldegeneration.

Human CNOT3 is an 18-exon gene that codes for a protein with 753 aminoacids. Exclusion of exon 2 removes the translation initiation codon fromthe transcript. Removal of exon 4, 5, 6, 7, 10 or 17 may disruptfunctional domains of CNOT3. Exclusion of any of exons 3, 8, 9, and11-16 disrupts the open reading frame.

Knocking out the CNOT3 gene is embryonically lethal, as it is vital forcell cycle progression through the regulation of mRNA turnover, and theregulation of mRNA decay in various physiological processes. However, ifthe levels of CNOT3 could be reduced or eliminated locally in the eye ofsubjects at risk of or suffering from RP, then the progression of thedisease would be affected as increased amounts of PRPF31 protein couldbe produced, mimicking an incomplete penetrance model whereinasymptomatic carriers are protected from the disease by a higher thanaverage expression of PRPF31 from an unmodified gene.

The present invention therefore provides antisense oligonucleotides toinduce non-productive splicing or functionally impaired protein of CNOT3(the negative regulator of PRPF31) to lower (but preferably not ablate)levels of CNOT3 and therefore increase transcription and translationfrom the normal PRPF31 allele.

In contrast to other antisense oligomer-based therapies, the presentinvention does not induce increased degradation of RNA via recruitmentof RNase H, wherein the RNase H preferentially binds and degraded RNAbound in duplex to DNA of the CNOT3 gene. Nor does it rely onhybridization of the antisense oligomer to the CNOT3 genomic DNA or thebinding of antisense oligomers to mRNA to modulate the amount of CNOT3protein produced by interfering with normal functions such asreplication, transcription, translocation and translation.

Rather, the antisense oligomers are used to modify pre-mRNA splicing ina CNOT3 gene transcript or part thereof and induce exon “skipping”and/or terminal intron retention. The strategy preferably reduces totalprotein expression or generates proteins which lack functional domains,leading to reduced protein function.

According to a first aspect of the invention, there is providedantisense oligomers capable of binding to a selected target on a CNOT3gene transcript to modify pre-mRNA splicing in a CNOT3 gene transcriptor part thereof. Broadly, there is provided an isolated or purifiedantisense oligomer for inducing targeted exon exclusion and/or terminalintron retention in a CNOT3 gene transcript or part thereof.

In the general population, expression of PRPF31 is highly variable, withlevels following a continuous distribution. Expression levels vary from0.53 to 2.48 arbitrary units, representing a 5-fold variation betweenlowest and highest expressors. Thirty percent of PRPF31 mutationcarriers have no symptoms and these individuals have wild-type allelesthat show almost 2-fold higher expression than those with symptomaticPRPF31 mutations. Therefore, preferably the modulation of CNOT3expression allows at least two-fold higher expression of PRPF31 thanthose with symptomatic PRPF31 mutations. Preferably the expression ofPRPF31 due to the AOs of the invention reducing the expression of CNOT3is between 1.5- and 5-fold higher than those with symptomatic PRPF31mutations. For example, the expression of PRPF31 may be between 2- and4-fold higher.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide” or “isolated oligonucleotide,” asused herein, may refer to a polynucleotide that has been purified orremoved from the sequences that flank it in a naturally-occurring state,e.g., a DNA fragment that is removed from the sequences that areadjacent to the fragment in the genome. The term “isolating” as itrelates to cells refers to the purification of cells (e.g., fibroblasts,lymphoblasts) from a source subject (e.g., a subject with apolynucleotide repeat disease). In the context of mRNA or protein,“isolating” refers to the recovery of mRNA or protein from a source,e.g., cells.

An antisense oligomer can be said to be “directed to” or “targetedagainst” a target sequence with which it hybridizes. In certainembodiments, the target sequence includes a region including a 3′ or 5′splice site of a pre-processed mRNA, a branch point, or other sequencesinvolved in the regulation of splicing. The target sequence may bewithin an exon or within an intron or spanning an intron/exon junction.

In certain embodiments, the antisense oligomer has sufficient sequencecomplementarity to a target RNA (i.e., the RNA for which splice siteselection is modulated) to block a region of a target RNA (e.g.,pre-mRNA) in an effective manner. In exemplary embodiments, suchblocking of CNOT3 pre-mRNA serves to modulate splicing, either bymasking a binding site for a native protein that would otherwisemodulate splicing and/or by altering the structure of the targeted RNA.In some embodiments, the target RNA is target pre-mRNA (e.g., CNOT3 genepre-mRNA).

An antisense oligomer having a sufficient sequence complementarity to atarget RNA sequence to modulate splicing of the target RNA means thatthe antisense oligomer has a sequence sufficient to trigger the maskingof a binding site for a native protein that would otherwise modulatesplicing and/or alters the three-dimensional structure of the targetedRNA.

Selected antisense oligomers can be made shorter, e.g., about 12 bases,or longer, e.g., about 50 bases, and include a small number ofmismatches, as long as the sequence is sufficiently complementary toeffect splice modulation upon hybridization to the target sequence, andoptionally forms with the RNA a heteroduplex having a Tm of 45° C. orgreater.

Preferably, the antisense oligomer is selected from the group comprisingthe sequences set forth in Table 1. Preferably, the antisense oligomeris selected from the group comprising the sequences in SEQ ID NOs: 1-74,more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64.

In certain embodiments, the degree of complementarity between the targetsequence and antisense oligomer is sufficient to form a stable duplex.The region of complementarity of the antisense oligomers with the targetRNA sequence may be as short as 8-11 bases, but can be 12-15 bases ormore, e.g., 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25bases, 12-20 bases, or 15-20 bases, including all integers in betweenthese ranges. An antisense oligomer of about 16-17 bases is generallylong enough to have a unique complementary sequence. In certainembodiments, a minimum length of complementary bases may be required toachieve the requisite binding Tm, as discussed herein.

In certain embodiments, oligonucleotides as long as 50 bases may besuitable, where at least a minimum number of bases, e.g., 10-12 bases,are complementary to the target sequence. In general, however,facilitated or active uptake in cells is optimized at oligonucleotidelengths of less than about 30 bases. For phosphorodiamidate morpholinooligomer (PMO) antisense oligomers, an optimum balance of bindingstability and uptake generally occurs at lengths of 18-25 bases.Included are antisense oligomers (e.g., CPPMOs, PPMOs, PMOs, PMO-X,PNAs, LNAs, 2′-OMe) that consist of about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases.

In certain embodiments, antisense oligomers may be 100% complementary tothe target sequence, or may include mismatches, e.g., to accommodatevariants, as long as a heteroduplex formed between the oligonucleotideand target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Hence, certain oligonucleotides may have about or at least about70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequencecomplementarity, between the oligonucleotide and the target sequence.

Mismatches, if present, are typically less destabilizing toward the endregions of the hybrid duplex than in the middle. The number ofmismatches allowed will depend on the length of the oligonucleotide, thepercentage of G:C base pairs in the duplex, and the position of themismatch(es) in the duplex, according to well understood principles ofduplex stability. Although such an antisense oligomer is not necessarily100% complementary to the target sequence, it is effective to stably andspecifically bind to the target sequence, such that splicing of thetarget pre-RNA is modulated.

The stability of the duplex formed between an antisense oligomer and atarget sequence is a function of the binding Tm and the susceptibilityof the duplex to cellular enzymatic cleavage. The Tm of anoligonucleotide with respect to complementary-sequence RNA may bemeasured by conventional methods, such as those described by Hames etal., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or asdescribed in Miyada C. G. and Wallace R. B., 1987, OligonucleotideHybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. Incertain embodiments, antisense oligomers may have a binding Tm, withrespect to a complementary-sequence RNA, of greater than bodytemperature and preferably greater than about 45° C. or 50° C. Tm's inthe range 60-80° C. or greater are also included.

Additional examples of variants include antisense oligomers having aboutor at least about 70% sequence identity or homology, e.g., 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity or homology, over the entire length of any of SEQID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27,30, 34, 35, 64 and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30, 34and 64 or the sequences provided in Table 1.

More specifically, there is provided an antisense oligomer capable ofbinding to a selected target site to modify pre-mRNA splicing in a CNOT3gene transcript or part thereof. The antisense oligomer is preferablyselected from those provided in Table 1 or SEQ ID NOs: 1-74, morepreferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67even more preferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64.

The modification of pre-mRNA splicing preferably induces “skipping”, orthe removal of one or more exons or introns of the mRNA and/or terminalintron retention. The resultant protein may be of a shorter length whencompared to the parent full-length CNOT3 protein due to either internaltruncation or premature termination or may be longer due to terminalintron retention. These CNOT3 proteins may be termed isoforms of theunmodified CNOT3 protein.

The remaining exons of the mRNA generated may be in-frame and produce ashorter protein with a sequence that is similar to that of the parentfull length protein, except that it has an internal truncation in aregion between the original 3′ and 5′ ends. In another possibility, theexon skipping may induce a frame shift that results in a protein whereinthe first part of the protein is substantially identical to the parentfull length protein, but wherein the second part of the protein has adifferent sequence (eg a nonsense sequence) due to a frame-shift.Alternatively, the exon skipping may induce the production of aprematurely terminated protein due to a disruption of the reading frameand presence of a premature termination of translation. Additionally,the antisense oligomer may produce an artificially lengthened protein,due to in-frame terminal intron retention.

Functional domains of CNOT3 include coiled coil, linker, NAR (Not anchorregion), CS (connector sequence) and the NOT box. Exclusion of exon 4removes part of the coiled coil domain, loss of exon 9 will disrupt theopen reading frame, leading to degradation of the mRNA and any encodedprotein will be truncated, whereas skipping exon 17 alters the NOT boxand is predicted to affect complex formation and therefore CNOT3function.

The removal of one or more exons may further lead to misfolding of theCNOT3 protein and a reduction in the ability of the protein to besuccessfully transported through the membrane.

The presence of internally truncated proteins (ie proteins lacking theamino acids encoded by one or more exons) is preferable. If the CNOT3protein is knocked out, there may be problems with elevation of CNOT3transcription as the body tries to compensate for the reduction in thetotal amount of CNOT3 protein. In contrast, the presence of aninternally truncated protein (preferably lacking one or more of thefeatures of the complete CNOT3 protein), should be sufficient to preventelevated transcription, but still provide a therapeutic advantage due toa reduction in the total amount of functional CNOT3 protein.

The antisense oligomer induced exon skipping of the present inventionneed not completely or even substantially ablate the function of theCNOT3 protein. Preferably, the exon skipping process results in areduced or compromised functionality of the CNOT3 protein.

The skipping process of the present invention, using antisenseoligomers, may skip an individual exon, or may result in skipping two ormore exons at once.

The antisense oligomers of the invention may be a combination of two ormore antisense oligomers capable of binding to a selected target toinduce exon exclusion in a CNOT3 gene transcript. The combination may bea cocktail of two or more antisense oligomers and/or a constructcomprising two or more antisense oligomers joined together.

TABLE 1 List of antisense oligonucleotide sequences used in thisstudy and efficacy score for AO-induced CNOT3 exon skipping.+1: >50% exon skipping, 0: <50% exon skipping, −1: No exon skippingSEQ ID JSR# Coordinates Sequence 5′−3′ 2−OMePS 1 4112 CNOT3_H2A(−6+19)ACUCUCUUGGAGACGGACGCUGCUA −1 2 4113 CNOT3_H2A(+28+52)AUCUUCCCUGCCCUACAGACGCACU −1 3 4114 CNOT3_H2D(+55−4)GUACCUUGGAGUUUGCGCUUGUCCG −1 4 3697 CNOT3_H3A(+9+33)CGGACACCUUCUUGAGGCAGCGAUC 1 5 3698 CNOT3_H3A(+39+63)GCCAAAUAUCUUCAAACUGCUCCAC 0 6 4579 CNOT3_H4A(−8+17)UUGGCUGCAUUGUGGAGCUGAGGGA −1 7 4580 CNOT3_H4A(+12+36)CUUUUCUUUCUGGUUCGCGUUGGCU 1 8 4581 CNOT3_H4A(+38+62)AUCUCCUUCUUUAGGUCAGCCUCAU 0 9 4582 CNOT3_H4D(+13−12)CAGCCCCCUCACUUGUAGCUUCUUA 1 10 4583 CNOT3_H5A(−9+16)UUUGGUCCCUCAGCCGCUGCAGAUG −1 11 4584 CNOT3_H5A(+36+60)CUGCCUCUUGUCCUUGAUCUCGUUG 1 12 4585 CNOT3_H5A(+53+77)UUGCGGUUGUCUAUAAGCUGCCUCU −1 13 4586 CNOT3_H5D(+13−12)CUGGGCUCCUACCGUCUCAAUGAGC −1 14 4587 CNOT3_H6A(−5+20)ACUUUGAACCGUUCCAUUUGCUGUA 1 15 4588 CNOT3_H6A(+12+36)GGUCUCUCGUUCCACAACUUUGAAC 0 16 4589 CNOT3_H6A(+37+61)CCUCUUUGCUGUAAGCUUUGGUUUU 1 17 4590 CNOT3_H6A(+89+113)ACCUCUUCCUUCUCCUUCUGGGCAG 1 18 4591 CNOT3_H6D(+15−10)CCCAACUCACCGUGAGCCACUGGCC 1 19 4592 CNOT3_H7A(−6+19)UGAGCGUGUCGAUGGUAUUCUAGGG −1 20 4593 CNOT3_H7A(+16+40)CAAACUGGUCCACCUGCAUGUUGAG −1 21 4594 CNOT3_H7A(+62+86)CCCUUCUUCUUGCGUGUCUGCACUG −1 22 4595 CNOT3_H7D(+15−10)CCUCACUCACAUCCUUGUCGCCCUU −1 23 3699 CNOT3_H8A(−10+15)CCGCTTCAAGCCCTCGCCCAGGGCC −1 24 3700 CNOT3_H8A(+18+41)GUGGUAGCGGUGCUUCUCGAUGUG −1 25 3701 CNOT3_H8A(+34+58)UGGUCUCUAGCAUGCGCACGUGGUA −1 26 3702 CNOT3_H8A(+75+99)GGCGUCAACGAGGAUGGAGUCAUUG 0 27 3703 CNOT3_H8A(+141+165)CUCCUCGAAGUCGGGGUCCUGGGAU 1 28 3704 CNOT3_H9A(−6+19)GUGGCGACCAGCGCCUGUGCUGUGG −1 29 3705 CNOT3_H9A(+29+52)CUCAUCCUCCAUGUGGCUGUGGCUG −1 30 3706 CNOT3_H9A(+56+80)GGGCGUGCUGCUGGACUGGUUGAAG 1 31 3707 CNOT3_H9A(+98+122)GGCUGGGCUGGGCGGGAUGGGAGAG 0 32 5485 CNOT3_H9A(+52+76)GUGCUGCUGGACUGGUUGAAGAUCU 0 33 4596 CNOT3_H10A(−10+15)AUCUUCAGAGUUUUCCUGAGGUAGG 1 34 4597 CNOT3_H10A(+16+40)CUGUGGAACGUCCCCUCUUCUUAUC 1 35 4598 CNOT3_H10D(+12−13)UCACACCCACCUGGCUGACUUCACU 1 36 4892 CNOT3_H11A(+49+73)AGGUGGGCGGCACAGCUGGGGACUG −1 37 4893 CNOT3_H11A(+56+80)GAGGGGUAGGUGGGCGGCACAGCUG −1 38 4894 CNOT3_H11A(+74+98)GCAGCAGGCGGGGGGCCGGAGGGGU −1 39 4115 CNOT3_H11A(−8+17)CCGUUUUUGGCUGGAGACUGCGGGU 0 40 4116 CNOT3_H11A(+171+195)GUGGCUGGGAGCUGGACUGGCCUUG 1 41 3708 CNOT3_H12A(+1+25)CUGUCUGCCACAACUGAGCUGUAAC NT 42 3709 CNOT3_H12A(+85+109)GGGUUGUGGGGGCCGGAAGGGGGGC NT 43 3710 CNOT3_H12D(+19−6)ACUCACGAGGUGCUGGGAGGUGGGU NT 44 4117 CNOT3_H13A(−6+19)UGCCGCACUGGGUUCCUUCCUGGAG 1 45 4118 CNOT3_H13A(+38+62)UGUUCCCUGAGCCUGGGGCCACGCC 0 46 4119 CNOT3_H13A(+83+107)GAGGAUUCACAGGCAGUGGCACCAG 1 47 4120 CNOT3_H14A(−8+17)CUCAGAGGCUCAGGGGCCUGGGGAG 0 48 4121 CNOT3_H14A(+48+72)AGGGUCCUCAAUGCCAGAGCUGAUG 0 49 4895 CNOT3_H15A(+147+171)GCAUGUGGUGCCAGGCGGCCUCUUC −1 50 4896 CNOT3_H15A(+157+181)GAGGGGUGAGGCAUGUGGUGCCAGG −1 51 4897 CNOT3_H15A(+175+199)CGAAUACGCUCAGAGUCAGAGGGGU −1 52 4122 CNOT3_H15A(−11+14)GCUCAGGAUGAUGUCUGUGGGGAGG −1 53 4123 CNOT3_H15A(+5+29)AGGUGCUGAUGUACUGCUCAGGAUG 1 54 4124 CNOT3_H15A(+42+66)CCUCUGACAGCUGCAGGGGCGGCUG 0 55 4125 CNOT3_H15A(+78+102)CCAUGGACAGACACCCAGCGACAGC 1 56 4126 CNOT3_H15A(+108+132)AGAGCUGCUCCUUGGUGAGGGGCAC 1 57 4127 CNOT3_H15A(+162+186)AGUCAGAGGGGUGAGGCAUGUGGUG 1 58 4128 CNOT3_H16A(−11+14)GGGGGAGGUACUGCCUGUGAGAGCA −1 59 4129 CNOT3_H16A(+36+58)GGUGGCAUCUGGUGGUGGUAGG 1 60 4130 CNOT3_H16A(+109+133)CUCCAGAUAGUAGAAGAUGAAGAAG 0 61 4365 CNOT3_H17A(+28+52)GCCAUGACUGCUUCUUUAGGGCCUU −1 63 4366 CNOT3_H17A(+57+81)GAACCACAUCAUGUACUUGGUGUGG −1 64 4367 CNOT3_H17A(+83+107)AUGGUCUUGGGCUCCUCGUGCCUCU 1 65 — CNOT3_H17A(+78+102)CUUGGGCUCCUCGUGCCUCUGGAAC NT 66 — CNOT3_H17A(+88+112)CAGUGAUGGUCUUGGGCUCCUCGUG NT 67 5486 CNOT3_H17A(+102+126)CUGCUCAAACUCGUCAGUGAUGGUC 0 68 3886 CNOT3_H18A(−10+15)GUAGAUGUAGGUGCCCUGGCCGGGG −1 69 3887 CNOT3_H18A(+16+40)GCUGGCCCCACUUCUCGUAGUCAAA 1 70 3888 CNOT3_H18A(+41+65)UCAAAGGUGAAGCCUUCCUUCUUCC 1 71 3889 CNOT3_H18A(+66+90)GUCCCGGUCCUCCAGGUAGCGGUAC −1 72 5551 CNOT3_H9A(+1+25)GGGGAGGUGGCGACCAGCGCCUGUG 0 73 5555 CNOT3_H17A(+71+95)UCCUCGUGCCUCUGGAACCACAUCA 1 74 4368 CNOT3_H17D(+15−10)GGGCCCUCACCUGCUCAAACUCGUC 0

There is also provided a method for manipulating splicing in a CNOT3gene transcript, the method including the step of:

-   -   a) providing one or more of the antisense oligomers as described        herein and allowing the oligomer(s) to bind to a target nucleic        acid site.

According to yet another aspect of the invention, there is provided asplice manipulation target nucleic acid sequence for CNOT3 comprisingthe DNA equivalents of the nucleic acid sequences selected from Table 1or the group consisting of SEQ ID NOs: 1-74, more preferably SEQ ID NOs:4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67 even more preferablySEQ ID NOs: 4, 7, 27, 30, 34 and 64, and sequences complementarythereto.

Designing antisense oligomers to completely mask consensus splice sitesmay not necessarily generate a change in splicing of the targeted exon.Furthermore, the inventors have discovered that size or length of theantisense oligomer itself is not always a primary factor when designingantisense oligomers. With some targets such as IGTA4 exon 3, antisenseoligomers as short as 20 bases were able to induce some exon skipping,in certain cases more efficiently than other longer (eg 25 bases)oligomers directed to the same exon.

The inventors have also discovered that there does not appear to be anystandard motif that can be blocked or masked by antisense oligomers toredirect splicing. It has been found that antisense oligomers must bedesigned, and their individual efficacy evaluated empirically.

More specifically, the antisense oligomer may be selected from those setforth in Table 1. The sequences are preferably selected from the groupconsisting of any one or more of any one or more of SEQ ID NOs: 1-74,more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64, andcombinations or cocktails thereof. This includes sequences which canhybridise to such sequences under stringent hybridisation conditions,sequences complementary thereto, sequences containing modified bases,modified backbones, and functional truncations or extensions thereofwhich possess or modulate pre-mRNA processing activity in a CNOT3 genetranscript.

The oligomer and the DNA, cDNA or RNA are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridisable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity or pairing such thatstable and specific binding occurs between the oligomer and the DNA,cDNA or RNA target. It is understood in the art that the sequence of anantisense oligomer need not be 100% complementary to that of its targetsequence to be specifically hybridisable. An antisense oligomer isspecifically hybridisable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA product, and there is a sufficient degree of complementarity toavoid non-specific binding of the antisense oligomer to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, and in the case of in vitro assays, underconditions in which the assays are performed.

Selective hybridisation may be under low, moderate or high stringencyconditions, but is preferably under high stringency. Those skilled inthe art will recognise that the stringency of hybridisation will beaffected by such conditions as salt concentration, temperature, ororganic solvents, in addition to the base composition, length of thecomplementary strands and the number of nucleotide base mismatchesbetween the hybridising nucleic acids. Stringent temperature conditionswill generally include temperatures in excess of 30° C., typically inexcess of 37° C., and preferably in excess of 45° C., preferably atleast 50° C., and typically 60° C.-80° C. or higher. Stringent saltconditions will ordinarily be less than 1000 mM, typically less than 500mM, and preferably less than 200 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. An example of stringent hybridisation conditions is 65° C.and 0.1×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate pH 7.0). Thus,the antisense oligomers of the present invention may include oligomersthat selectively hybridise to the sequences provided in Table 1, or SEQID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27,30, 34, 35, 64 and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30, 34and 64.

It will be appreciated that the codon arrangements at the end of exonsin structural proteins may not always break at the end of a codon,consequently there may be a need to delete more than one exon from thepre-mRNA to ensure in-frame reading of the mRNA. In such circumstances,a plurality of antisense oligomers may need to be selected by the methodof the invention wherein each is directed to a different regionresponsible for inducing inclusion of the desired exon and/or intron. Ata given ionic strength and pH, the Tm is the temperature at which 50% ofa target sequence hybridizes to a complementary polynucleotide. Suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

Typically, selective hybridisation will occur when there is at leastabout 55% identity over a stretch of at least about 14 nucleotides,preferably at least about 65%, more preferably at least about 75% andmost preferably at least about 90%, 95%, 98% or 99% identity with thenucleotides of the antisense oligomer. The length of homologycomparison, as described, may be over longer stretches and in certainembodiments will often be over a stretch of at least about ninenucleotides, usually at least about 12 nucleotides, more usually atleast about 20, often at least about 21, 22, 23 or 24 nucleotides, atleast about 25, 26, 27 or 28 nucleotides, at least about 29, 30, 31 or32 nucleotides, at least about 36 or more nucleotides.

Thus, the antisense oligomer sequences of the invention preferably haveat least 75%, more preferably at least 85%, more preferably at least 86,87, 88, 89 or 90% homology to the sequences shown in the sequencelistings herein. More preferably there is at least 91, 92, 93 94, or95%, more preferably at least 96, 97, 98% or 99%, homology. Generally,the shorter the length of the antisense oligomer, the greater thehomology required to obtain selective hybridisation. Consequently, wherean antisense oligomer of the invention consists of less than about 30nucleotides, it is preferred that the percentage identity is greaterthan 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95%, 96, 97, 98% or 99% compared with the antisense oligomers setout in the sequence listings herein. Nucleotide homology comparisons maybe conducted by sequence comparison programs such as the GCG WisconsinBestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research12, 387-395). In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The antisense oligomer of the present invention may have regions ofreduced homology, and regions of exact homology with the targetsequence. It is not necessary for an oligomer to have exact homology forits entire length. For example, the oligomer may have continuousstretches of at least 4 or 5 bases that are identical to the targetsequence, preferably continuous stretches of at least 6 or 7 bases thatare identical to the target sequence, more preferably continuousstretches of at least 8 or 9 bases that are identical to the targetsequence. The oligomer may have stretches of at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 bases that areidentical to the target sequence. The remaining stretches of oligomersequence may be intermittently identical with the target sequence; forexample, the remaining sequence may have an identical base, followed bya non-identical base, followed by an identical base. Alternatively (oras well) the oligomer sequence may have several stretches of identicalsequence (for example 3, 4, 5 or 6 bases) interspersed with stretches ofless than perfect homology. Such sequence mismatches will preferablyhave no or very little loss of splice switching activity.

The term “modulate” or “modulates” includes to “increase” or “decrease”one or more quantifiable parameters, optionally by a defined and/orstatistically significant amount. The terms “increase” or “increasing,”“enhance” or “enhancing,” or “stimulate” or “stimulating” refergenerally to the ability of one or antisense oligomers or compositionsto produce or cause a greater physiological response (i.e., downstreameffects) in a cell or a subject relative to the response caused byeither no antisense oligomer or a control compound. The terms“decreasing” or “decrease” refer generally to the ability of one orantisense oligomers or compositions to produce or cause a reducedphysiological response (i.e., downstream effects) in a cell or a subjectrelative to the response caused by either no antisense oligomer or acontrol compound.

Relevant physiological or cellular responses (in vivo or in vitro) willbe apparent to persons skilled in the art, and may include increases inthe exclusion of specific exons in a CNOT3-coding pre-mRNA, decreases inthe amount of CNOT3-coding pre-mRNA or decreases in the expression offunctional CNOT3 protein in a cell, tissue, or subject in need thereof.An “decreased” or “reduced” amount is typically a statisticallysignificant amount, and may include a decrease that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000times) (including all integers and decimal points in between and above1, e.g., 1.5, 1.6, 1.7. 1.8) less than the amount produced when noantisense oligomer is present (the absence of an agent) or a controlcompound is used.

The term “reduce” or “inhibit” may relate generally to the ability ofone or more antisense oligomers or compositions to “decrease” a relevantphysiological or cellular response, such as a symptom of a disease orcondition described herein, as measured according to routine techniquesin the diagnostic art. Relevant physiological or cellular responses (invivo or in vitro) will be apparent to persons skilled in the art, andmay include reductions in the symptoms or pathology of a disease such asretinitis pigmentosa.

A “decrease” in a response may be statistically significant as comparedto the response produced by no antisense oligomer or a controlcomposition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease,including all integers in between.

The length of an antisense oligomer may vary, as long as it is capableof binding selectively to the intended location within the pre-mRNAmolecule. The length of such sequences can be determined in accordancewith selection procedures described herein. Generally, the antisenseoligomer will be from about 10 nucleotides in length, up to about 50nucleotides in length. It will be appreciated, however, that any lengthof nucleotides within this range may be used in the method. Preferably,the length of the antisense oligomer is between 10 and 40, 10 and 35, 15to 30 nucleotides in length or 20 to 30 nucleotides in length, mostpreferably about 25 to 30 nucleotides in length. For example, theoligomer may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotidesin length.

As used herein, an “antisense oligomer” refers to a linear sequence ofnucleotides, or nucleotide analogs, that allows the nucleobase tohybridize to a target sequence in an RNA by Watson-Crick base pairing,to form an oligonucleotide:RNA heteroduplex within the target sequence.The terms “antisense oligomer”, “antisense oligonucleotide”, “oligomer”and “antisense compound” may be used interchangeably to refer to anoligonucleotide. The cyclic subunits may be based on ribose or anotherpentose sugar or, in certain embodiments, a morpholino group (seedescription of morpholino oligonucleotides below). Also contemplated arepeptide nucleic acids (PNAs), locked nucleic acids (LNAs), and2′-O-Methyl oligonucleotides, among other antisense agents known in theart.

Included are non-naturally-occurring antisense oligomers, or“oligonucleotide analogs”, including antisense oligomers oroligonucleotides having (i) a modified backbone structure, e.g., abackbone other than the standard phosphodiester linkage found innaturally-occurring oligo- and polynucleotides, and/or (ii) modifiedsugar moieties, e.g., morpholino moieties rather than ribose ordeoxyribose moieties. Oligonucleotide analogs support bases capable ofhydrogen bonding by Watson-Crick base pairing to standard polynucleotidebases, where the analog backbone presents the bases in a manner topermit such hydrogen bonding in a sequence-specific fashion between theoligonucleotide analog molecule and bases in a standard polynucleotide(e.g., single-stranded RNA or single-stranded DNA). Preferred analogsare those having a substantially uncharged, phosphorus containingbackbone.

One method for producing antisense oligomers is the methylation of the2′ hydroxyribose position and the incorporation of a phosphorothioatebackbone produces molecules that superficially resemble RNA but that aremuch more resistant to nuclease degradation, although persons skilled inthe art of the invention will be aware of other forms of suitablebackbones that may be useable in the objectives of the invention.

To avoid degradation of pre-mRNA during duplex formation with theantisense oligomers, the antisense oligomers used in the method may beadapted to minimise or prevent cleavage by endogenous RNase H. Thisproperty is highly preferred, as the treatment of the RNA with theunmethylated oligomers, either intracellular or in crude extracts thatcontain RNase H, leads to degradation of the pre-mRNA:antisense oligomerduplexes. Any form of modified antisense oligomers that is capable ofby-passing or not inducing such degradation may be used in the presentmethod. The nuclease resistance may be achieved by modifying theantisense oligomers of the invention so that it comprises partiallyunsaturated aliphatic hydrocarbon chain and one or more polar or chargedgroups including carboxylic acid groups, ester groups, and alcoholgroups.

Antisense oligomers that do not activate RNase H can be made inaccordance with known techniques (see, e.g., U.S. Pat. No. 5,149,797).Such antisense oligomers, which may be deoxyribonucleotide orribonucleotide sequences, simply contain any structural modificationwhich sterically hinders or prevents binding of RNase H to a duplexmolecule containing the oligomer as one member thereof, which structuralmodification does not substantially hinder or disrupt duplex formation.Because the portions of the oligomer involved in duplex formation aresubstantially different from those portions involved in RNase H bindingthereto, numerous antisense oligomers that do not activate RNase H areavailable. For example, such antisense oligomers may be oligomerswherein at least one, or all, of the inter-nucleotide bridging phosphateresidues are modified phosphates, such as methyl phosphonates, methylphosphorothioates, phosphoromorpholidates, phosphoropiperazidatesboranophosphates, amide linkages and phosphoramidates. For example,every other one of the internucleotide bridging phosphate residues maybe modified as described. In another non-limiting example, suchantisense oligomers are molecules wherein at least one, or all, of thenucleotides contain a 2′ lower alkyl moiety (such as, for example,C₁-C₄, linear or branched, saturated or unsaturated alkyl, such asmethyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).For example, every other one of the nucleotides may be modified asdescribed.

An example of antisense oligomers which when duplexed with RNA are notcleaved by cellular RNase H is 2′-O-methyl derivatives. Such2′-O-methyl-oligoribonucleotides are stable in a cellular environmentand in animal tissues, and their duplexes with RNA have higher Tm valuesthan their ribo- or deoxyribo-counterparts. Alternatively, the nucleaseresistant antisense oligomers of the invention may have at least one ofthe last 3′-terminus nucleotides fluoridated. Still alternatively, thenuclease resistant antisense oligomers of the invention havephosphorothioate bonds linking between at least two of the last3-terminus nucleotide bases, preferably having phosphorothioate bondslinking between the last four 3′-terminal nucleotide bases.

Increased splice-switching may also be achieved with alternativeoligonucleotide chemistry. For example, the antisense oligomer may bechosen from the list comprising: phosphoramidate or phosphorodiamidatemorpholino oligomer (PMO); PMO-X; PPMO; peptide nucleic acid (PNA); alocked nucleic acid (LNA) and derivatives including alpha-L-LNA,2′-amino LNA, 4′-methyl LNA and 4′-O-methyl LNA; ethylene bridgednucleic acids (ENA) and their derivatives; phosphorothioate oligomer;tricyclo-DNA oligomer (tcDNA); tricyclophosphorothioate oligomer;2′O-Methyl-modified oligomer (2′-OMe); 2′-O-methoxy ethyl (2′-MOE);2′-fluoro, 2′-fluroarabino (FANA); unlocked nucleic acid (UNA); hexitolnucleic acid (HNA); cyclohexenyl nucleic acid (CeNA); 2′-amino (2′-NH2);2′-O-ethyleneamine or any combination of the foregoing as mixmers or asgapmers. To further improve the delivery efficacy, the above-mentionedmodified nucleotides are often conjugated with fattyacids/lipid/cholesterol/aminoacids/carbohydrates/polysaccharides/nanoparticles etc. to the sugar ornucleobase moieties. These conjugated nucleotide derivatives can also beused to construct exon skipping antisense oligomers. Antisenseoligomer-induced splice modification of the human CNOT3 gene transcriptshave generally used either oligoribonucleotides, PNAs, 2OMe or MOEmodified bases on a phosphorothioate backbone. Although 2OMeAOs are usedfor oligo design, due to their efficient uptake in vitro when deliveredas cationic lipoplexes, these compounds are susceptible to nucleasedegradation and are not considered ideal for in vivo or clinicalapplications. When alternative chemistries are used to generate theantisense oligomers of the present invention, the uracil (U) of thesequences provided herein may be replaced by a thymine (T).

While the antisense oligomers described above are a preferred form ofthe antisense oligomers of the present invention, the present inventionincludes other oligomeric antisense molecules, including but not limitedto oligomer mimetics such as are described below.

Specific examples of preferred antisense oligomers useful in thisinvention include oligomers containing modified backbones or non-naturalinter-nucleoside linkages. As defined in this specification, oligomershaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified oligomers that do not have a phosphorusatom in their inter-nucleoside backbone can also be considered to beantisense oligomers.

In other preferred oligomer mimetics, both the sugar and theinter-nucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligomer mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligomer isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleo-bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

Another preferred chemistry is the phosphorodiamidate morpholinooligomer (PMO) oligomeric compounds, which are not degraded by any knownnuclease or protease. These compounds are uncharged, do not activateRNase H activity when bound to a RNA strand and have been shown to exertsustained splice modulation after in vivo administration (Summerton andWeller, Antisense Nucleic Acid Drug Development, 7, 187-197).

Modified oligomers may also contain one or more substituted sugarmoieties. Oligomers may also include nucleobase (often referred to inthe art simply as “base”) modifications or substitutions. Certainnucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C., even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligomers of the invention involveschemically linking to the oligomer one or more moieties or conjugatesthat enhance the activity, cellular distribution or cellular uptake ofthe oligomer. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,myristyl, or an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety.

Cell penetrating peptides have been added to phosphorodiamidatemorpholino oligomers to enhance cellular uptake and nuclearlocalization. Different peptide tags have been shown to influenceefficiency of uptake and target tissue specificity, as shown inJearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624-1629.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligomer. The present invention alsoincludes antisense oligomers that are chimeric compounds. “Chimeric”antisense oligomers or “chimeras,” in the context of this invention, areantisense oligomers, particularly oligomers, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligomer compound. These oligomerstypically contain at least one region wherein the oligomer is modifiedso as to confer upon the oligomer or antisense oligomer increasedresistance to nuclease degradation, increased cellular uptake, and anadditional region for increased binding affinity for the target nucleicacid.

The activity of antisense oligomers and variants thereof can be assayedaccording to routine techniques in the art. For example, splice formsand expression levels of surveyed RNAs and proteins may be assessed byany of a wide variety of well-known methods for detecting splice formsand/or expression of a transcribed nucleic acid or protein. Non-limitingexamples of such methods include RT-PCR of spliced forms of RNA followedby size separation of PCR products, nucleic acid hybridization methodse.g., Northern blots and/or use of nucleic acid arrays; nucleic acidamplification methods; immunological methods for detection of proteins;protein purification methods; and protein function or activity assays.

RNA expression levels can be assessed by preparing mRNA/cDNA (i.e., atranscribed polynucleotide) from a cell, tissue or organism, and byhybridizing the mRNA/cDNA with a reference polynucleotide, which is acomplement of the assayed nucleic acid, or a fragment thereof. cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction or in vitro transcription methods prior to hybridization withthe complementary polynucleotide; preferably, it is not amplified.Expression of one or more transcripts can also be detected usingquantitative PCR to assess the level of expression of the transcript(s).

The present invention provides antisense oligomer inducedsplice-switching of the CNOT3 gene transcript, clinically relevantoligomer chemistries and delivery systems to direct CNOT3 splicemanipulation to therapeutic levels. Substantial decreases in the amountof full length CNOT3 mRNA, and hence CNOT3 protein from CNOT3 genetranscription, are achieved by:

-   1) oligomer refinement in vitro using fibroblast cell lines, through    experimental assessment of (i) intronic-enhancer target motifs, (ii)    antisense oligomer length and development of oligomer    cocktails, (iii) choice of chemistry, and (iv) the addition of    cell-penetrating peptides (CPP) to enhance oligomer delivery; and-   2) detailed evaluation of a novel approach to generate CNOT3    transcripts with one or more missing exons.

As such, it is demonstrated herein that processing of CNOT3 pre-mRNA canbe manipulated with specific antisense oligomers. In this wayfunctionally significant decreases in the amount of CNOT3 protein can beobtained, thereby reducing the severe pathology associated withretinitis pigmentosa.

The antisense oligomers used in accordance with this invention may beconveniently made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Onemethod for synthesising oligomers on a modified solid support isdescribed in U.S. Pat. No. 4,458,066.

Any other means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligomers such as the phosphorothioates and alkylatedderivatives. In one such automated embodiment, diethyl-phosphoramiditesare used as starting materials and may be synthesized as described byBeaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

The antisense oligomers of the invention are synthesised in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisenseoligomers. The molecules of the invention may also be mixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules etc.

The antisense oligomers may be formulated for oral, topical, parenteralor other delivery, particularly formulations for topical ocular andinjectable ocular delivery. The formulations may be formulated forassisting in uptake, distribution and/or absorption at the site ofdelivery or activity. Preferably the antisense oligomers of the presentinvention are formulated for delivered topically to the eye or byintraocular injection or intraocular implant, so that the effects onCNOT3 production are spatially limited and are not systemic.

Method of Treatment

According to a still further aspect of the invention, there is providedone or more antisense oligomers as described herein for use in anantisense oligomer-based therapy. Preferably, the therapy is for acondition related to CNOT3 expression. More preferably, the therapy fora condition related to CNOT3 expression is therapy for retinitispigmentosa.

More specifically, the antisense oligomer may be selected from Table 1,or the group consisting of any one or more of SEQ ID NOs: 1-74, morepreferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67even more preferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64, andcombinations or cocktails thereof. This includes sequences which canhybridise to such sequences under stringent hybridisation conditions,sequences complementary thereto, sequences containing modified bases,modified backbones, and functional truncations or extensions thereofwhich possess or modulate pre-mRNA processing activity in a CNOT3 genetranscript.

The invention extends also to a combination of two or more antisenseoligomers capable of binding to a selected target to induce exonexclusion in a CNOT3 gene transcript. The combination may be a cocktailof two or more antisense oligomers, a construct comprising two or moreor two or more antisense oligomers joined together for use in anantisense oligomer-based therapy.

There is therefore provided a method to treat, prevent or ameliorate theeffects of a disease associated with CNOT3 expression, comprising thestep of:

-   -   a) administering to the patient an effective amount of one or        more antisense oligomers or pharmaceutical composition        comprising one or more antisense oligomers as described herein.

Preferably the disease associated with CNOT3 expression in a patient isretinitis pigmentosa.

Therefore, the invention provides a method to treat, prevent orameliorate the effects of retinitis pigmentosa, comprising the step of:

-   -   a) administering to the patient an effective amount of one or        more antisense oligomers or pharmaceutical composition        comprising one or more antisense oligomers as described herein.

Preferably, the therapy is used to reduce the levels of functional CNOT4protein via an exon skipping strategy. The reduction in levels of CNOT3is preferably achieved by reducing the transcripts level throughmodifying pre-mRNA splicing in the CNOT3 gene transcript or partthereof.

The reduction in CNOT3 will preferably lead to a reduction in thequantity, duration or severity of the symptoms of a CNOT3-relatedcondition or pathology, such as retinitis pigmentosa.

As used herein, “treatment” of a subject (e.g. a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

The subject with the disease associated with CNOT3 expression may be amammal, including a human.

The antisense oligomers of the present invention may also be used inconjunction with alternative therapies, such as drug therapies.

The present invention therefore provides a method of treating,preventing or ameliorating the effects of a disease or conditionassociated with CNOT3 expression, wherein the antisense oligomers of thepresent invention and administered sequentially or concurrently withanother alternative therapy associated with treating, preventing orameliorating the effects of a disease or condition associated with CNOT3expression. Preferably, the disease or condition is retinitispigmentosa.

Delivery

The antisense oligomers of the present invention also can be used as aprophylactic or therapeutic, which may be utilised for the purpose oftreatment of a disease. Accordingly, in one embodiment the presentinvention provides antisense oligomers that bind to a selected target inthe CNOT3 pre-mRNA to induce efficient and consistent exon skipping asdescribed herein, in a therapeutically effective amount, admixed with apharmaceutically acceptable carrier, diluent, or excipient.

There is also provided a pharmaceutical, prophylactic, or therapeuticcomposition to treat, prevent or ameliorate the effects of a diseaserelated to CNOT3 expression in a patient, the composition comprising:

a) one or more antisense oligomers as described herein; and

b) one or more pharmaceutically acceptable carriers and/or diluents.

Preferably, the antisense oligomer of the present invention is deliveredvia a localised ocular route to avoid a systemic effect. Routes ofadministration include, but are not limited to, intravitreal,intracameral, subconjunctival, subtenon, retrobulbar, posteriorjuxtascleral, or topical (drops, eye washes, creams etc). Deliverymethods include, for example, injection by a syringe and a drug deliverydevice, such as an implanted vitreal delivery device (i.e., VITRASERT®).

In one embodiment, the antisense oligomer is administered intravenouslyat a dose of 20 mg/kg. For example, the antisense oligomer may beadministered intravenously at a dose of 20 mg/kg in a mouse.

Preferably, the antisense oligomer is administered via intravitrealinjection at between 0.01-1.5 mg/kg body weight, between 0.1-0.1 mg/kgbody weight, between 0.2-0.8 mg/kg body weight, between 0.4-0.7 mg/kgbody weight, or more preferably between 0.4-0.6 mg/kg body weight. Theantisense oligomer may be administered via intravitreal injection at,for example, about 0.05 mg/kg body weight, 0.1 mg/kg body weight, 0.2mg/kg body weight, 0.3 mg/kg body weight, 0.4 mg/kg body weight, 0.5mg/kg body weight, 0.6 mg/kg body weight, 0.7 mg/kg body weight, 0.8mg/kg body weight, 0.9 mg/kg body weight, 1.0 mg/kg body weight, 1.1mg/kg body weight, 1.2 mg/kg body weight, 1.3 mg/kg body weight, 1.4mg/kg body weight, 1.5 mg/kg body weight. Preferably, the antisenseoligomer is administered via intravitreal injection at about 0.5 mg/kgbody weight. For example, the antisense oligomer may be administered viaintravitreal injection at about 0.5 mg/kg body weight to a mouse.

More preferably, the antisense oligomer is administered via intravitrealinjection at between 0.5-50 mg per eye, 0.5-40 mg per eye, 0.5-30 mg pereye, 2-30 mg per eye, 2-20 mg per eye, 0.5-20 mg per eye, or morepreferably between 5-20 mg per eye. The antisense oligomer may beadministered via intravitreal injection at, for example, about 0.5 mgper eye, 1.0 mg per eye, 2.0 mg per eye, 3.0 mg per eye, 4.0 mg per eye,5.0 mg per eye, 6.0 mg per eye, 7.0 mg per eye, 8.0 mg per eye, 9.0 mgper eye, 10.0 mg per eye, 11.0 mg per eye, 12.0 mg per eye, 13.0 mg pereye, 14.0 mg per eye, 15.0 mg per eye, 16.0 mg per eye, 17.0 mg per eye,18.0 mg per eye, 19.0 mg per eye, 20.0 mg per eye, 21 mg per eye, 22 mgper eye, 23 mg per eye, 24 mg per eye, 25 mg per eye, 30 mg per eye, 35mg per eye, 40 mg per eye, 45 mg per eye, or 50 mg per eye. Preferably,the antisense oligomer is administered via intravitreal injection atabout 5-20 mg per eye.

The antisense oligomer may be administered at regular intervals for ashort time period, e.g., daily for two weeks or less. However, in manycases the oligomer is administered intermittently over a longer periodof time. Administration may be followed by, or concurrent with,administration of an antibiotic or other therapeutic treatment. Thetreatment regimen may be adjusted (dose, frequency, route, etc.) asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

Dosing may be dependent on severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Alternatively, dosing may be titratedagainst disease progression rate. A baseline progression is established.Then the progression rate after an initial once off dose is monitored tocheck that there is a reduction in the rate. Preferably, there is noprogression after dosing. Preferably, re-dosing is only necessary ifprogression rate is unchanged. Successful treatment preferably resultsin no further progression of the disease or even some recovery ofvision. Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates.

Optimum dosages may vary depending on the relative potency of individualoligomers, and can generally be estimated based on EC50s found to beeffective in in vitro and in vivo animal models.

In general, dosage is from 0.01-1.5 mg/kg body weight or administrationvia intravitreal injection at between 0.5-50 mg per eye and may be givenonce or more daily, weekly, monthly or yearly, or even once every 2 to20 years. Repetition rates for dosing depend on progression rate of thedisease. Persons of ordinary skill in the art can easily estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligomer is administered in maintenance doses, the dose maybe 0.01-1.5 mg/kg body weight or administration via intravitrealinjection at between 0.5-50 mg per eye, once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years.

An effective in vivo treatment regimen using the antisense oligomers ofthe invention may vary according to the duration, dose, frequency androute of administration, as well as the condition of the subject undertreatment (i.e., prophylactic administration versus administration inresponse to localized or systemic infection). Accordingly, such in vivotherapy will often require monitoring by tests appropriate to theparticular type of disorder under treatment, and correspondingadjustments in the dose or treatment regimen, in order to achieve anoptimal therapeutic outcome.

Treatment may be monitored, e.g., by general indicators of disease knownin the art. The efficacy of an in vivo administered antisense oligomersof the invention may be determined from biological samples (tissue,blood, urine etc.) taken from a subject prior to, during and subsequentto administration of the antisense oligomer. Assays of such samplesinclude (1) monitoring the presence or absence of heteroduplex formationwith target and non-target sequences, using procedures known to thoseskilled in the art, e.g., an electrophoretic gel mobility assay; (2)monitoring the amount of a mutant mRNA in relation to a reference normalmRNA or protein as determined by standard techniques such as RT-PCR,Northern blotting, ELISA or Western blotting.

Intranuclear oligomer delivery is a major challenge for antisenseoligomers. Different cell-penetrating peptides (CPP) localize PMOs tovarying degrees in different conditions and cell lines, and novel CPPshave been evaluated by the inventors for their ability to deliver PMOsto the target cells. The terms CPP or “a peptide moiety which enhancescellular uptake” are used interchangeably and refer to cationic cellpenetrating peptides, also called “transport peptides”, “carrierpeptides”, or “peptide transduction domains”. The peptides, as shownherein, have the capability of inducing cell penetration within about orat least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of agiven cell culture population and allow macromolecular translocationwithin multiple tissues in vivo upon systemic administration. CPPs arewell-known in the art and are disclosed, for example in U.S. ApplicationNo. 2010/0016215, which is incorporated by reference in its entirety.

The present invention therefore provides antisense oligomers of thepresent invention win combination with cell-penetrating peptides formanufacturing therapeutic pharmaceutical compositions.

Excipients

The antisense oligomers of the present invention are preferablydelivered in a pharmaceutically acceptable composition. The compositionmay comprise about 1 nM to 1000 nM of each of the desired antisenseoligomer(s) of the invention. Preferably, the composition may compriseabout 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 10 nM to 500 nM,1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20nM, most preferably between 1 nM and 10 nM of each of the antisenseoligomer(s) of the invention.

The composition may comprise about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7nm, 8 nm, 9 nm, 10 nm, 20 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or 1000 nm of each of thedesired antisense oligomer(s) of the invention.

The present invention further provides one or more antisense oligomersadapted to aid in the prophylactic or therapeutic treatment, preventionor amelioration of symptoms of a disease such as an CNOT3 expressionrelated disease or pathology in a form suitable for delivery to apatient.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similarly untoward reaction, such as gastricupset and the like, when administered to a patient. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thecompound is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Remington: The Science and Practice of Pharmacy, 22ndEd., Pharmaceutical Press, PA (2013).

In a more specific form of the invention there are providedpharmaceutical compositions comprising therapeutically effective amountsof one or more antisense oligomers of the invention together withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants, and/or carriers. Such compositions includediluents of various buffer content (e.g. Tris-HCl, acetate, phosphate),pH and ionic strength and additives such as detergents and solubilizingagents (e.g. Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g. Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). The materialmay be incorporated into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hylauronic acid may also be used. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, for example,Remington: The Science and Practice of Pharmacy, 22nd Ed.,Pharmaceutical Press, PA (2013). The compositions may be prepared inliquid form, or may be in dried powder, such as a lyophilised form.

It will be appreciated that pharmaceutical compositions providedaccording to the present invention may be administered by any meansknown in the art. The pharmaceutical compositions for administration areadministered by injection, orally, topically or by the pulmonary ornasal route. For example, the antisense oligomers may be delivered byintravenous, intra-arterial, intraperitoneal, intramuscular orsubcutaneous routes of administration. The appropriate route may bedetermined by one of skill in the art, as appropriate to the conditionof the subject under treatment. Preferably, the antisense oligomers aredelivered topically to the eye or by intraocular injection orintraocular implant, so that the effects on CNOT3 production arespatially limited and are not systemic.

Formulations for topical administration include those in which theoligomers of the disclosure are in admixture with a topical deliveryagent such as lipids, liposomes, fatty acids, fatty acid esters,steroids, chelating agents and surfactants. Lipids and liposomes includeneutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other administration, oligomers ofthe disclosure may be encapsulated within liposomes or may formcomplexes thereto, in particular to cationic liposomes. Alternatively,oligomers may be complexed to lipids, in particular to cationic lipids.Fatty acids and esters, pharmaceutically acceptable salts thereof, andtheir uses are further described in U.S. Pat. No. 6,287,860 and/or U.S.patent application Ser. No. 09/315,298 filed on May 20, 1999.

In certain embodiments, the antisense oligomers of the disclosure can bedelivered by topical or transdermal methods (e.g., via incorporation ofthe antisense oligomers into, e.g., emulsions, with such antisenseoligomers optionally packaged into liposomes) including delivery toocular surfaces. Such topical or transdermal andemulsion/liposome-mediated methods of delivery are described fordelivery of antisense oligomers in the art, e.g., in U.S. Pat. No.6,965,025. Preferably the topical delivery is delivery to the eye.

The antisense oligomers described herein may also be delivered via animplantable device. Design of such a device is an art-recognizedprocess, with, e.g., synthetic implant design described in, e.g., U.S.Pat. No. 6,969,400. Preferably the implant is able to be implanted intothe eye for sustained delivery of the antisense oligomers.

Compositions and formulations for ocular administration, includingocular injection, topical ocular delivery and ocular implant may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients.

The delivery of a therapeutically useful amount of antisense oligomersmay be achieved by methods previously published. For example, deliveryof the antisense oligomer may be via a composition comprising anadmixture of the antisense oligomer and an effective amount of a blockcopolymer. An example of this method is described in US patentapplication US20040248833. Other methods of delivery of antisenseoligomers to the nucleus are described in Mann C J et al. (2001) Proc,Natl. Acad. Science, 98(1) 42-47, and in Gebski et al. (2003) HumanMolecular Genetics, 12(15): 1801-1811. A method for introducing anucleic acid molecule into a cell by way of an expression vector eitheras naked DNA or complexed to lipid carriers, is described in U.S. Pat.No. 6,806,084.

Antisense oligomers can be introduced into cells using art-recognizedtechniques (e.g., transfection, electroporation, fusion, liposomes,colloidal polymeric particles and viral and non-viral vectors as well asother means known in the art). The method of delivery selected willdepend at least on the cells to be treated and the location of the cellsand will be apparent to the skilled artisan. For instance, localizationcan be achieved by liposomes with specific markers on the surface todirect the liposome, direct injection into tissue containing targetcells, specific receptor-mediated uptake, or the like.

It may be desirable to deliver the antisense oligomer in a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes or liposome formulations. These colloidal dispersion systemscan be used in the manufacture of therapeutic pharmaceuticalcompositions.

Liposomes are artificial membrane vesicles, which are useful as deliveryvehicles in vitro and in vivo. These formulations may have net cationic,anionic, or neutral charge characteristics and have usefulcharacteristics for in vitro, in vivo and ex vivo delivery methods. Ithas been shown that large unilamellar vesicles can encapsulate asubstantial percentage of an aqueous buffer containing largemacromolecules. RNA and DNA can be encapsulated within the aqueousinterior and be delivered to cells in a biologically active form(Fraley, et al., Trends Biochem. Sci. 6:77, 1981).

In order for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of theantisense oligomer of interest at high efficiency while not compromisingtheir biological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988). Thecomposition of the liposome is usually a combination of phospholipids,particularly high phase-transition-temperature phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations. Cationic liposomes are positively charged liposomes which arebelieved to interact with negatively charged DNA molecules to form astable complex. Liposomes that are pH-sensitive or negatively-chargedare believed to entrap DNA rather than complex with it. Both cationicand noncationic liposomes have been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860.

As known in the art, antisense oligomers may be delivered using, forexample, methods involving liposome-mediated uptake, lipid conjugates,polylysine-mediated uptake, nanoparticle-mediated uptake, andreceptor-mediated endocytosis, as well as additional non-endocytic modesof delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (refer to Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44, 35-49, incorporated by reference inits entirety).

The antisense oligomer may also be combined with other pharmaceuticallyacceptable carriers or diluents to produce a pharmaceutical composition.Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The composition may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular, oral,or transdermal administration.

The routes of administration described are intended only as a guidesince a skilled practitioner will be able to readily determine theoptimum route of administration and any dosage for any particular animaland condition.

Multiple approaches for introducing functional new genetic material intocells, both in vitro and in vivo have been attempted (Friedmann (1989)Science, 244:1275-1280). These approaches include integration of thegene to be expressed into modified retroviruses (Friedmann (1989) supra;Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S);integration into non-retrovirus vectors (Rosenfeld, et al. (1992) Cell,68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or deliveryof a transgene linked to a heterologous promoter-enhancer element vialiposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med.Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288;Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; andWang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855);coupled to ligand-specific, cation-based transport systems (Wu and Wu(1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA,expression vectors (Nabel et al. (1990), supra); Wolff et al. (1990)Science, 247:1465-1468). Direct injection of transgenes into tissueproduces only localized expression (Rosenfeld (1992) supra); Rosenfeldet al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra;and Hazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med.Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) havereported in vivo transfection only of lungs of mice following eitherintravenous or intratracheal administration of a DNA liposome complex.An example of a review article of human gene therapy procedures is:Anderson, Science (1992) 256:808-813; Barteau et al. (2008), Curr GeneTher; 8(5):313-23; Mueller et al. (2008). Clin Rev Allergy Immunol;35(3):164-78; Li et al. (2006) Gene Ther., 13(18):1313-9; Simoes et al.(2005) Expert Opin Drug Deliv; 2(2):237-54.

The antisense oligomers of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, as an example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such pro-drugs,and other bioequivalents.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e. salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligomers, preferred examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be via topical (including ophthalmic and mucousmembranes, as well as rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer,intratracheal, intranasal, epidermal and transdermal), oral orparenteral routes. Parenteral administration includes intravenous,intra-arterial, subcutaneous, intraperitoneal, intraocular orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Oligomers with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor intraocular administration. Preferably, the antisense oligomer isdelivered via the intraocular route.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Swiss-Style

According to another aspect of the invention there is provided the useof one or more antisense oligomers as described herein in themanufacture of a medicament for the modulation or control of a diseaseassociated with CNOT3 expression.

The invention also provides for the use of purified and isolatedantisense oligomers as described herein, for the manufacture of amedicament for treatment of a disease associated with CNOT3 expression.

There is also provided the use of purified and isolated antisenseoligomers as described herein, for the manufacture of a medicament totreat, prevent or ameliorate the effects of a disease associated withCNOT3 expression.

Preferably, the CNOT3-related pathology or disease is retinitispigmentosa.

The invention extends, according to a still further aspect thereof, tocDNA or cloned copies of the antisense oligomer sequences of theinvention, as well as to vectors containing the antisense oligomersequences of the invention. The invention extends further also to cellscontaining such sequences and/or vectors.

Kits

There is also provided a kit to treat, prevent or ameliorate the effectsof a disease associated with CNOT3 expression in a patient, which kitcomprises at least an antisense oligomer as described herein andcombinations or cocktails thereof, packaged in a suitable container,together with instructions for its use.

In a preferred embodiment, the kits will contain at least one antisenseoligomer as described herein or as shown in Table 1, or SEQ ID NOs:1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34,35, 64 and 67 even more preferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64or a cocktail of antisense oligomers, as described herein. The kits mayalso contain peripheral reagents such as buffers, stabilizers, etc.

There is therefore provided a kit to treat, prevent or ameliorate adisease or condition associated with CNOT3 expression in a patient,which kit comprises at least an antisense oligomer described herein oras shown in Table 1 and combinations or cocktails thereof, packaged in asuitable container, together with instructions for its use.

There is also provided a kit to treat, prevent or ameliorate a diseaseor condition associated with CNOT3 expression in a patient, which kitcomprises at least an antisense oligomer selected from the groupconsisting of any one or more of SEQ ID NOs: 1-74, more preferably SEQID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67 even morepreferably SEQ ID NOs: 4, 7, 27, 30, 34 and 64, and combinations orcocktails thereof, packaged in a suitable container, together withinstructions for its use.

Preferably, the disease or condition is retinitis pigmentosa.

The contents of the kit can be lyophilized and the kit can additionallycontain a suitable solvent for reconstitution of the lyophilizedcomponents. Individual components of the kit would be packaged inseparate containers and, associated with such containers, can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. For in vivo use, the expression construct maybe formulated into a pharmaceutically acceptable syringeablecomposition. In this case the container means may itself be an inhalant,syringe, pipette, eye dropper, or other such like apparatus, from whichthe formulation may be applied to an affected area of the animal, suchas the lungs, injected into an animal, or even applied to and mixed withthe other components of the kit.

In an embodiment, the kit of the present invention comprises acomposition comprising a therapeutically effective amount of anantisense oligomer capable of binding to a selected target on a CNOT3gene transcript to modify pre-mRNA splicing in a CNOT3 gene transcriptor part thereof. In an alternative embodiment, the formulation is inpre-measured, pre-mixed and/or pre-packaged. Preferably, the intraocularsolution is sterile.

The kit of the present invention may also include instructions designedto facilitate user compliance. Instructions, as used herein, refers toany label, insert, etc., and may be positioned on one or more surfacesof the packaging material, or the instructions may be provided on aseparate sheet, or any combination thereof. For example, in anembodiment, the kit of the present invention comprises instructions foradministering the formulations of the present invention. In oneembodiment, the instructions indicate that the formulation of thepresent invention is suitable for the treatment of retinitis pigmentosa.Such instructions may also include instructions on dosage, as well asinstructions for administration via topical delivery to the eye or viaintraocular injection.

The antisense oligomers and suitable excipients can be packagedindividually so to allow a practitioner or user to formulate thecomponents into a pharmaceutically acceptable composition as needed.Alternatively, the antisense oligomers and suitable excipients can bepackaged together, thereby requiring de minimus formulation by thepractitioner or user. In any event, the packaging should maintainchemical, physical, and aesthetic integrity of the active ingredients.

General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. The invention includes all such variation andmodifications. The invention also includes all of the steps, features,formulations and compounds referred to or indicated in thespecification, individually or collectively and any and all combinationsor any two or more of the steps or features.

Each document, reference, patent application or patent cited in thistext is expressly incorporated herein in their entirety by reference,which means that it should be read and considered by the reader as partof this text. That the document, reference, patent application or patentcited in this text is not repeated in this text is merely for reasons ofconciseness.

Any manufacturer's instructions, descriptions, product specifications,and product sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention.

The present invention is not to be limited in scope by any of thespecific embodiments described herein. These embodiments are intendedfor the purpose of exemplification only. Functionally equivalentproducts, formulations and methods are clearly within the scope of theinvention as described herein.

The invention described herein may include one or more range of values(eg. Size, displacement and field strength etc). A range of values willbe understood to include all values within the range, including thevalues defining the range, and values adjacent to the range which leadto the same or substantially the same outcome as the values immediatelyadjacent to that value which defines the boundary to the range.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. Hence “about 80%” means “about 80%” and also “80%”.At the very least, each numerical parameter should be construed in lightof the number of significant digits and ordinary rounding approaches.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the invention.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs. The term “active agent” may meanone active agent, or may encompass two or more active agents.

Sequence identity numbers (“SEQ ID NO:”) containing nucleotide and aminoacid sequence information included in this specification are collectedat the end of the description and have been prepared using the programpatent In Version 3.0. Each nucleotide or amino acid sequence isidentified in the sequence listing by the numeric indicator <210>followed by the sequence identifier (e.g. <210>1, <210>2, etc.). Thelength, type of sequence and source organism for each nucleotide oramino acid sequence are indicated by information provided in the numericindicator fields <211>, <212> and <213>, respectively. Nucleotide andamino acid sequences referred to in the specification are defined by theinformation provided in numeric indicator field <400> followed by thesequence identifier (e.g. <400>1, <400>2, etc.).

An antisense oligomer nomenclature system was proposed and published todistinguish between the different antisense oligomers (see Mann et al.,(2002) J Gen Med 4, 644-654). This nomenclature became especiallyrelevant when testing several slightly different antisense oligomers,all directed at the same target region, as shown below:

-   -   H # A/D (x:y)    -   the first letter designates the species (e.g. H: human, M:        murine)    -   “#” designates target exon number    -   “A/D” indicates acceptor or donor splice site at the beginning        and end of the exon, respectively    -   (x y) represents the annealing coordinates where “−” or “+”        indicate intronic or exonic sequences respectively. As an        example, A(−6+18) would indicate the last 6 bases of the intron        preceding the target exon and the first 18 bases of the target        exon. The closest splice site would be the acceptor so these        coordinates would be preceded with an “A”. Describing annealing        coordinates at the donor splice site could be D(+2−18) where the        last 2 exonic bases and the first 18 intronic bases correspond        to the annealing site of the antisense oligomer. Entirely exonic        annealing coordinates that would be represented by A(+65+85),        that is the site between the 65th and 85th nucleotide,        inclusive, from the start of that exon.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these methods in no way serve to limit the true scope ofthis invention, but rather are presented for illustrative purposes.

EXAMPLES Example 1 AO Mediated Exon Skipping to Induce a Frame-Shift inCNOT3

The exon structure of CNOT3 is shown in FIG. 1. Splice switching AOswere designed to target enhancer sites within frame-shifting exons inCNOT3 pre-mRNA (FIG. 1), to induce exon skipping and consequently, lossof the open reading frame and knockdown of CNOT3.

Normal human fibroblasts and fibroblasts from an RP11 patient (PRPF31c.1205 C>A Ser402*) were obtained from skin biopsies and cultured inDMEM supplemented with 10% FBS. Antisense sequences (Table 1),synthesised in-house as 2′O-Methyl phosphorothioate oligomers weretransfected into fibroblasts in 24 well plates as lipoplexes usingLipofectamine 3000 (Life Technologies), as per the manufacturer'sinstructions. Sequences that were effective in altering target exonselection were then synthesized as phosphorodiamidate morpholinooligomers (PMO) and transfected into fibroblasts as i) uncomplexed ii)annealed to a sense ODN leash and delivered with Lipofectamine 3000 (asabove), or iii) nucleofection using the P2 Primary cell 4-D NucleofectorX kit S (Lonza), as per manufacturer's instructions.

Total RNA was extracted from the transfected and control cells using theMagMax RNA extraction system (Life Technologies). Transcripts ofinterest are assessed by semi-quantitative RT-PCR in the first instance,and then by qRT-PCR. cDNA (RNA 300 ng) was synthesised, as permanufacturer's instructions, using SuperScript IV reverse transcriptasekit (Life Technologies). RT-PCRs were done using LA Taq DNA polymerasewith GC buffer I (TAKARA), as per the manufacturer's instructions.

Three different primer sets were used to assess CNOT3 transcripts aftercells were transfected with the CNOT3 exon skipping AOs:

-   -   1. For AOs targeting exon 3, a forward primer annealing to exon        2 was paired with a reverse primer in exon 6.    -   2. For AOs targeting exons 8 and 9, a forward primer in exon 7        was paired with an exon 11 reverse primer.    -   3. For AOs targeting exons 16 and 17, a forward primer in exon        15 was paired with a reverse primer in exon 18.

qRT-PCR was used to quantitate PRPF31 transcript levels aftertransfection with the CNOT3 exon skipping AOs. A forward primer acrossthe PRPF31 exon 2 and 3 junction was paired with a reverse primer acrossthe exon 3 and 4 junction (Table 2). Results for all CNOT3 AO treatedcells were normalised to the expression of two housekeeping genes, TBPand GAPDH, and compared to Anti ISS-N1 and sham AO treated cells inorder to calculate fold-changes in PRPF31 transcripts.

TABLE 2Primers for RT-PCR and qRT-PCR of CNOT3, PRPF31 and housekeeping genes,TBP and GAPDH. SEQ ID NO Gene Primer name Primer sequence 5′-3′ Conc.Analysis 75 CNOT3 Exon 2 Forward GAAGATGGCGGACAAGCGCAA  50 nM RT-PCR 76CNOT3 Exon 6 Reverse CTGGCCAACCTCTTCCTTCTC  50 nM RT-PCR 77 CNOT3Exon 7 Forward CTGTCAGTGCAGACACGCAA  50 nM RT-PCR 78 CNOT3Exon 11 Reverse GGACAGGCTGGAGCCGTTT  50 nM RT-PCR 79 CNOT3Exon 15 Forward CATCCTGAGCAGTACATCAGC  50 nM RT-PCR 80 CNOT3Exon 18 Reverse CTCCAGGTAGCGGTACTCAA  50 nM RT-PCR 81 PRPF31Exon 2/3 Forward GGGATAGTAAGATGTTTGCTGAG 250 nM qRT-PCR 82 PRPF31Exon 3/4 Reverse GTCCCATCACTTCTGAAGCTTTGG 250 nM qRT-PCR 83 TBP FTCAGGCGTTCGGTGGATCGAGT 500 nM qRT-PCR 84 TBP R AGTGATGCTGGGCACTGCGGAGAA500 nM qRT-PCR 85 GAPDH F ACAGTCAGCCGCATCTTCTT 250 nM qRT-PCR 86 GAPDH RAGGGGTCTACATGGCAACTG 250 nM qRT-PCR 87 CNOT3 Exon 10 ForwardGACGTTCCACAGACAGTGAAG 88 CNOT3 Exon 8 Reverse GTGGTAGCGGTGCTTCTCGATG 89CNOT3 3_5′UTR Forward TATATTCGGGACTCGGGGG

Sequencing of the induced (smaller) CNOT3 transcript products, predictedto result from induced exon skipping revealed that AOs 3697 and 3698mediated exon 3 skipping, AO 3702 induced both partial and completeskipping of exon 8 and AO 3703 mediated dose dependent exon 8 skipping(data not shown). The AOs that mediated efficient CNOT3 exon 3, 8 or 9skipping were then transfected into RP patient fibroblasts for 48 hrsand CNOT3 transcripts were analysed by RT-PCR (FIG. 2). CNOT3 exonskipping was evident 48 hr after transfection, with some reduction inthe levels of full-length transcript product. Exon 3 was excluded by AO3697, exon 8 was skipped by AOs 3702 and 3703 and exon 9 by AO 3706.

Previously, we have shown that the phosphorodiamidate morpholino (PMO)chemistry more effectively mediates splice modification than the samesequences synthesized as the 2′O-Methyl PS chemistry. Therefore, themost promising AO sequences for CNOT3 transcript knockdown will besynthesised as PMOs and transfected into normal fibroblasts uncomplexed,with a leash/lipoplex or via nucleofection (Nucleofector, Lonza).CNOT3-targeting AO sequence evaluation and optimisation is ongoing.

SH-SY5Y cells are often used as in vitro models of neuronal function anddifferentiation. They are adrenergic in phenotype but also expressdopaminergic markers. AOs that modify CNOT3 transcripts in fibroblastswill be evaluated in differentiated SH-SY5Y cells for the ability todown-regulate CNOT3 and increase PRPF31 expression (transcript andprotein).

Example 2 AO Mediated Terminal Intron Retention to Knock Down CNOT3

Antisense sequences were designed to target the terminal exon of CNOT3in order to induce terminal intron retention and knock-down of CNOT3.

2′O-Methyl AOs targeting the terminal exon of CNOT3 were transfectedinto adRP11 patient fibroblasts for 48 hr. RT-PCR analysis of the CNOT3transcripts showed AOs 3887, 3888 and 3889 (Table 1) induced terminalintron retention and reduction in the level of full-length transcriptproduct, in a dose dependent manner (FIG. 3).

Example 3

Recruitment and Review of Families with RP

We have examined 3 generations of 2 families in WA (1 Caucasian and 1Aboriginal) with PRPF31 mutations (FIG. 4). We have obtained dermalfibroblast from 7 patients and have started monitoring diseaseprogression in 10 of 24 affected individuals in these families.

TABLE 3 RP11-causing mutations identified and respective number ofpatients with each confirmed mutation (information from AustralianInherited retinal disease register as of 2018). Mutation Protein changePhenotype Number affected (state) PRPF31.1205C > A Ser402* Dominant RP 9(WA) PRPF31.267delA Glu89Aspfs Dominant RP 8 (WA, Vic) PRPF31.527 + 3A >G N/A: SPLICE Dominant RP 3 (NSW) PRPF31.319C > G Leu107Val Dominant RP1 (SA) PRPF31.527 + 1G > T N/A: SPLICE Dominant RP 1 (WA)PRPF31.1289_1290insA X Dominant RP 1 (SA)

Example 4

Generate Induced Pluripotent Stem Cells from RP11 Patient and ControlFibroblasts and Assess Gene Expression as a Consequence of CNOT3Knockdown

Induced pluripotent stem cells will be generated from patient andcontrol fibroblasts. Patient fibroblasts will be transfected withreprogramming episomes (ThermoFisher®) using the NEON® electroporationsystem. In a typical reprogramming experiment, 12-15 iPSC-like coloniesare picked 3-4 weeks after transfection and subcultured for evaluationof pluripotent gene expression by immunostaining and RT-PCR analysis(FIG. 5A-C). Three clones will then be selected for additional testing,including gene expression profiling by TaqMan Arrays (Human Stem CellPluripotency Array, ThermoFisher) and virtual karyotyping by chromosomalG-band analyses with QuantiSNP analysis (AGRF).

To demonstrate tri-lineage differentiation potential, iPSC are culturedas embryoid bodies for 2-4 weeks and examined for expression of markersof ectoderm, mesoderm and endoderm differentiation, as well as thedownregulation of pluripotency markers by RT-PCR (FIG. 5C). The iPSCswill be differentiated to retinal organoids using CIF's publishedprotocol (Mellough et al., Efficient stage-specific differentiation ofhuman pluripotent stem cells toward retinal photoreceptor cells. StemCells, 2012. 30(4): p. 673-86), (FIG. 5D-H).

The differentiated cells will be transfected with the leadCNOT3-targeting AO, synthesised as a morpholino compound, in triplicatefor each analysis.

CNOT3, PRPF31 and other splicing factor transcripts will be analysed.CNOT3, PRPF31 and selected paraspeckle proteins and splicing factorswill be assessed by Western blot and immunofluorescence to reflect theintegrity of the splicing machinery and pathways.

Example 5 Dose Dependent Testing of AOs

AO sequences were designed to skip selected exons from the CNOT3messenger RNA and transfected as 20-Methyl phosphorothioate AOs intofibroblasts after complexing with cationic liposomes for efficienttransfection. Skipping of the target exon results in a shortenedmessenger RNA RT-PCR product, identified by separation and staining ofthe products on a 2% agarose gel (FIGS. 6 and 7).

Preferred sequences show dose dependent target exon skipping. The leadsequences were then synthesized as phosphorodiamidate morpholinooligomers (PPMO) for testing by transfection into patient fibroblasts.

Patients with RP11 show PRPF31 levels of approximately 50% of those inthe healthy population. Messenger RNAs for CNOT3 and PRPF31 werequantified by reverse transcription and quantitative PCR (qRT-PCR) in RP11 family members and in a healthy control. FIG. 8a shows that higherCNOT3 expression correlates with reduced PRPF31 and clinical outcome inheterozygous PRPF31 mutation carriers. Skipping of target CNOT3 exonswas assessed by qRT-PCR (FIG. 8b, 8c ), and the PRPF31 expression afterAO treatment is shown relative to that in cells treated with a controlAO sequence (control AO 25 nM transfection value set to 1) that does nottarget any region of the healthy human genome (no effectexpected—negative control). Data from untreated cells is included forcomparison.

Patients with RP11 show PRPF31 levels of approximately 50% of those inthe healthy population, whereas asymptomatic PRPF31 mutation carriersshow levels of at least 70% of healthy levels, or higher. An increase of1.5-fold of PRPF31 expression (e.g. CNOT3 exon 3 or 10 skipping, FIG. 8band CNOT3 exons 9, 16, or 17 skipping, FIG. 8c ) is expected to rescuesplicing function in RP11 retinal pigment epithelium.

Example 6

Antisense Oligomer-Mediated CNOT3 Exon Skipping Upregulates PRPF31Expression in RP11 Patient iPSC-Derived Retinal Pigment Epithelium (RPE)and Rescues Primary Cilia Length and Number

ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17) wassynthesized as phosphorodiamidate morpholino oligomer (PMO) andtransfected into RP11 iPSC-derived RPE by direct transfection using 5 uMASO in culture media.

Immunocytochemistry was used for immunostaining of cilia and basal bodyin wildtype and RP11 RPE with or without antisense oligomer treatment.

Cilia Immunostaining Protocol

RPE cells on a chamber slide were fixed using ice cold acetone-methanol(1:1) for 4 minutes then air dried. Cells were blocked for 30 minutes in10% filtered goat serum in PBS. For basal body staining, cells wereincubated with mouse anti-pericentrin antibody (1:100) for 1 hr at roomtemperature. Primary antibody was detected using AlexaFluoro488anti-mouse (1:400). For cilia staining, cells were incubated with rabbitanti-ARL13B antibody (1:500) and incubated overnight at 4° C. Secondprimary antibody was detected using AlexaFluoro568 anti-rabbit (1:400)for 1 hr at room temperature and counterstained with Hoechst for nucleidetection (dilution 1:125). A coverslip was mounted onto a slide usingProlong Gold anti-fade media.

Confocal Analysis and Quantification

Primary cilia of RPE were assessed to evaluate PRPF31 function usingconfocal microscopy. Approximately 300 RPE cells/sample were measuredfor cilia length using NIS-Elements Imaging Software.

1. An isolated or purified antisense oligomer for modifying pre-mRNAsplicing in the CNOT3 gene transcript or part thereof.
 2. The antisenseoligomer of claim 1 that induces non-productive splicing or functionalimpairment in the CNOT3 gene transcript or part thereof.
 3. Theantisense oligomer of claim 1 selected from the list comprising: SEQ IDNOs: 1-74.
 4. The antisense oligomer of claim 1 wherein the antisenseoligomer contains one or more nucleotide positions subject to analternative chemistry or modification chosen from the list comprising:(i) a modified backbone structure; (ii) modified sugar moieties; (iii)resistance to RNase H; (iv) oligomeric mimetic chemistry.
 5. Theantisense oligomer of claim 1 wherein the antisense oligomer is furthermodified by: (i) chemical conjugation to a moiety; and/or (ii) taggingwith a cell penetrating peptide.
 6. The antisense oligomer of claim 1wherein the antisense oligomer is a phosphorodiamidate morpholinooligomer.
 7. The antisense oligomer of claim 1 wherein when any uracil(U) is present in the nucleotide sequence, the uracil (U) is replaced bya thymine (T).
 8. The antisense oligomer of claim 1 that operates toinduce skipping of one or more of the exons of the CNOT3 gene transcriptor part thereof.
 9. The antisense oligomer of claim 1 that results inthe expression of a PRPF31 protein between 1.5 and 5 fold higher thanthe expression of the PRPF31 protein in subjects with symptomatic PRPF31mutations.
 10. A method for manipulating splicing in a CNOT3 genetranscript, the method including the step of: a) providing one or moreof the antisense oligomers according to claim 1 and allowing theoligomer(s) to bind to a target nucleic acid site.
 11. A pharmaceutical,prophylactic, or therapeutic composition to treat, prevent or amelioratethe effects of a disease related to CNOT3 expression in a patient, thecomposition comprising: a) one or more antisense oligomers according toclaim 1, and b) one or more pharmaceutically acceptable carriers and/ordiluents.
 12. A method to treat, prevent or ameliorate the effects of adisease associated with CNOT3 expression, comprising the step of: a)administering to the patient an effective amount of one or moreantisense oligomers or pharmaceutical composition comprising one or moreantisense oligomers according to claim
 1. 13. (canceled)
 14. A kit totreat, prevent or ameliorate the effects of a disease associated withCNOT3 expression in a patient, which kit comprises at least an antisenseoligomer according to claim 1, packaged in a suitable container,together with instructions for its use.
 15. The method of claim 12,wherein the CNOT3 expression related disease is retinitis pigmentosa.16. The method of claim 12 wherein the subject with the diseaseassociated with CNOT3 expression is a human.
 17. The method of claim 12that results in the expression of a PRPF31 protein between 1.5 and 5fold higher than the expression of the PRPF31 protein in subjects withsymptomatic PRPF31 mutations.
 18. The antisense oligomer of claim 1selected from the list comprising: SEQ ID NOs: 4, 7, 9, 11, 15, 16-18,27, 30, 34, 35 and
 64. 19. The antisense oligomer of claim 1 selectedfrom the list comprising: SEQ ID NOs: 4, 7, 27, 30, 34 and 64.